Preparation of monoglycerides of butyrate

EXAMPLE 1
The esterification reaction takes place in batches of 10,000 kg.
3000 kg of butyric acid and 7000 kg of glycerol are introduced into a reactor at ambient temperature.
The temperature is increased to 14O0C, the butyric acid that evaporates being recycled within the reactor by means of a
reflux condenser.
The further raising of the temperature from 140 to 17O 0C must be very slow (over about 4 hours) and the reflux condenser
temperature must be maintained at 12O0C in order to evaporate the water derived from the esterification reaction while
the butyric acid continues to recycle within the reactor.
At this point the temperature can be raised to 180 0C (but leaving the reflux condenser temperature at 12O 0C) and once
this temperature has been reached the acidity of the mixture is expected to reach a value less than 1 %. A vacuum is then
applied to distil off any unreacted butyric acid until a final acidity of less than 0.2% is reached.
The mixture is discharged through a cooler to bring it to ambient temperature. A mixture is thus obtained containing 43%
monoglyceride ester, 6% diglyceride ester, 1% triglyceride ester, and 50% glycerol.
Once the esterification reaction is complete the glycerol can be separated if desired by distillation from the thus obtained
mono- di- and triglyceride esters to arrive at a 90% monoglyceride concentration.
EXAMPLE 2
Sixty 5 week old DanBred piglets were assigned to two groups of thirty piglets each: A) - control, and B) treated, divided
into 6 pens of ten animals each. After the first 10 days of adaptation in the enclosures, all animals were inoculated orally
with Salmonella typhimurium, isolated at the lstituto Zooprofilattico of Forli (Italy) from fecal samples of infected pigs, with
a dose equal to 7 x 107 cfu. The following day some of the subjects from each pen presented with diarrhoea. The
symptoms worsened and affected all the subjects over the next three days following infection.
Fecal samples were collected on the third day following infection; the bacterial count was found to be equal to 165,000 cfu
in control group A) and 160,000 cfu in the treated group B). Group B) from the third day after infection was treated with a
mixture composed of: - Butyric acid monoglycerides = 45%
- Butyric acid diglycerides = 6%
- Butyric acid triglycerides = 1%
- Glycerol = 48% administered in the drinking water at a dosage of 0.5% for three days. On the third day after treatment,
fecal samples were again collected for bacterial count analysis. The control group A) presented a mean cfu number of
160,000, while in the treated group B) the cfu number was 900. Use of the "butyric acid esters and glycerol" mixture in the
stated percentages reduced the cfus of salmonella by 3
Iog10, with a 3-day administration. This fact confirms the bactericidal effectiveness of the mixture.
EXAMPLE 3: The present field trial was carried out on an Italian farm with hygiene problems such as very evident ileitis
resulting from a Lawsonia intracellular'^ infection, enteritis from Brachyspira Spp and necrotic enteritis resulting from a
Treponema hyodysenteriae infection. 1,027 DanBred pigs weighing about 25 kg (71 days old) were divided into two
groups: control group A) and treated group B)1 composed of 511 and 516 animals respectively.

The two groups were fed with a feed that was formulated in identical manner except for the following components: the
feed of the control group had added Lincomycin, 200 ppm, and Doxicyclin, 250 ppm, for the first 14 days of the trial, and
Lincomycin alone for the remaining time. The treated group B) did not receive antibiotics in the feed, only a "butyric acid
esters and glycerol" mixture composed as follows:
- Butyric acid monoglycerides = 45%
- Butyric acid diglycerides - 6% - Butyric acid triglycerides = 1%
- Glycerol = 48% administered to the feed in a quantity of 0.5% to replace 0.5% of the soya oil. The trial lasted 63 days.
The growth and feeding efficiency results are summarized in the table below. Table

Although the fecal analysis of the control group A) showed the presence of Lawsonia, its presence was not found in the
treated group B). The diarrhoea episodes were also very much reduced in the treated group B). The growth parameters,
the feed conversion index of the treated group B) were comparable, and tendentially better than those of the control group
A) whose diet contained the aforesaid antibiotics. The "butyric acid esters and glycerol" mixture enabled the highlighted
diseases to be controlled, without the use of antibiotics. The trial has demonstrated the antibacterial effect of the "butyric
acid esters and glycerol" mixture with a consequent improvement to intestinal health.
EXAMPLE 4

Efficacy test towards Salmonella typhimurium in chickens

Salmonella strain For the test, a strain of Salmonella typhimurium isolated and identified by the
IZSLER section of Foril was used.
Animals
SPF (Specific Pathogen Free) chicks were used, 30 animals per test. The chicks were hatched at the I2SLER section of
Forli. The subjects were immediately placed into isolation units.
Diet
The animals received water from the mains water supply and a commercial starter ad libitum feed. The feed contained
added Monobutyrin 43.
Experimental protocol 4 groups of 30 subjects each were prepared. The diets differed by the different amount of
Monobtyrin 43 added to the feed from the first day of life, and were identified as follows: untreated control group: 0%,
group 1: 1% in the feed, group 2: 0.3% in the feed. Group 3 received the same feed as the control group up to the 14 th day
of life, i.e. until the 7th day post-infection, and only received feed supplemented with 1.4% Monobutyri 43 after that day.
At aged 7 days, all the subjects were infected by the esophageal route with 10 7 cfu of Salmonella typhimurium. 24 hours
following infection, cloacal swabs were taken from all the subjects to confirm that Salmonella typhimurium infection had
taken hold. At 14, 24 and 35 days of life, 10 subjects in each group were killed. The ceca were collected from each animal
and the load of Salmonella typhimurium was determined (expressed in cfu/g). Laboratory tests
The absence of antibodies against S. typhimurium was confirmed by an ELISA test. The cloacal swabs were seeded
directly onto Hektoen Enteric Agar and incubated at 370C for 24 hours. One gram of intestinal contents was diluted in 9 ml
of Ringer's lactate and seeded onto Hektoen Enteric Agar (inoculum volume: 0.1 ml). Colony counting was carried out
after 24 hours of incubation at 37C. For each collection, the geometric means of the bacterial loads of the 10 killed
subjects were calculated. All the subjects, after one day of life, were found to be seronegative for Salmonella typhimurium.
24 hours after the infection, all the cloacal swabs were found to be positive for S. typhimurium. The results of the
determined cecal bacterial loads are shown in the following table. Table CFU in the cecum of chickens infected with
Salmonella Typhimurium - 107

EXAMPLE 5
In vitro sensitivity tests towards filamentous fungi (moulds)

Materials and methods

Strains of Aspergillus spp, Penicillium spp and Fusarium spp were utilized for the test, having been isolated and identified
during diagnostic activity at the IZSLER section of Forll from complete feeds used in the chicken industry. To prepare the
inoculum, mycelium of pure cultures of the tested strains was collected using a swab. The material thus collected was
dissolved in a culture broth (BHI - Brain Heart Infusion). 5 ml of the fungal suspension and an equal amount of the product
to be tested were placed in contact in a test tube. The test tube was incubated at 20+4 0C for 24 hours.
After this time period, the fungal suspension was then seeded and enumerated. The control suspension was obtained by
placing 5 ml of fungal suspension + 5 ml of diluent (Ringer's lactate) into a test tube. Reading of the tests was carried out
after a 5 day incubation period at 20 40G.
The results given in the following table are expressed as cfu/ml
Table

Note: Monopropionin 43 is composed of. 43% propionic acid monoglycerides 12% propionic acid diglycerides 1 %
propionic acid triglycerides 28% free glycerol 16% H2O
Note: Moobutyrin 43 is composed of: 43% butyric acid monoglycerides 6% butyric acid diglycerides 1 % butyric acid
triglycerides 50% glycerol
EXAMPLE 6
In vitro efficacy test towards Penicilium spp and Fusarium spp Materials and methods
Strains: strains of moulds isolated and identified by the IZSLER section at Forll were used for the test. The strains were
revitalized in BHI broth then enumerated in OGYE agar (after incubation at 20 0C for 5 days) Substrate: a complete
chicken feed, sterilized in a dry oven at 1000C for 4 hours, was used.
Efficacy test: 10 g of feed were inoculated with 2 ml of fungal suspension (in distilled water) to which 70 l of the product
to be tested was added. The mixture thus obtained was kept at ambient temperature. A positive control (infected and

untreated) and a negative control (feed only + distilled water) were also prepared. On days 7 and 14 following infection,
the fungal concentrations in the treated sample and control samples were evaluated. The results are given in the table
below.
TABLE

Note: Monopropionin 43 is composed of: 43% propionic acid monoglycerides 12% propionic acid diglycerides 1 %
propionic acid triglycerides 28% free glycerol 16% H2O